1. Field of the Invention
The present invention relates to a monoclonal antibody or an antibody fragment thereof, which specifically recognizes three-dimensional structure of an extracellular region of a polypeptide encoded by Claudin-4 (hereinafter referred to as “CLDN4”) gene and binds to the extracellular region; a monoclonal antibody or an antibody fragment thereof, which specifically recognizes three-dimensional structures of extracellular regions of a polypeptide encoded by a Claudin-3 (hereinafter referred to as “CLDN3”) gene and a polypeptide encoded by a CLDN4 gene, and binds to both of the extracellular regions; a hybridoma which produces the antibody; a DNA which encodes the antibody; a vector which comprises the DNA; a transformant obtained by transforming the vector; a process for producing an antibody or an antibody fragment thereof using the hybridoma or the transformant; and a diagnostic agent or a therapeutic agent using the antibody or the antibody fragment thereof.
2. Brief Description of the Background Art
As the Claudin molecules (hereinafter Claudin is referred to as “CLDN”), 24 kinds of family molecules have so far been reported, and it is considered that CLDN molecules are homophilicly or heterophilicly bound to each other and function in intercellular adhesion (Non-patent Document 1). It is considered that a tight junction (hereinafter referred to as “TJ”) formed by CLDN functions as a barrier for preventing free pass of water-soluble molecules through the gap between epithelial cells or vascular endothelial cells (Non-patent Documents 1 and 4). Also, it is considered that TJ also functions as a partition of membrane protein and lipid molecules between the apex and lateral base of cell membrane and as a fence to block their mutual diffusion (Non-patent Document 1).
CLDN4 is a four-transmembrane protein containing total 209 amino acids in length and is an adhesion molecule which constitutes TJ, cloned in 1997 by Katahira et al. as a receptor for Welch bacillus enterotoxin [Clostridium perfringens enterotoxin (hereinafter referred to as “CPE”)] (Non-patent Documents 2 and 3).
Regarding expression of CLDN4 in cancer, its high level expression has been reported in various cancers such as pancreatic cancer (Non-patent Documents 5, 6 and 7), ovarian cancer (Non-patent Documents 8 and 9), breast cancer (Non-patent Document 10), uterine cancer (Non-patent Document 11), colorectal cancer (Non-patent Document 12), stomach cancer (Non-patent Documents 13 and 14), prostate cancer (Non-patent Document 15), esophageal carcinoma (Non-patent Document 16) and biliary cancer (Non-patent Document 17), and it has been reported that high expression of CLDN4 is a cause of poor prognosis in stomach cancer (Non-patent Document 13). Regarding the activity of CLDN4 in cancers, it has been reported that tumor forming ability and cell moving ability are accelerated in a CLDN4-expressed cancer cell and that the infiltrating ability of a cancer cell is reduced when expression of CLDN4 in the cell is suppressed using a small interfering RNA for CLDN4 (Non-patent Document 18).
On the other hand, similar to CLDN4, Claudin-3 (hereinafter referred to as “CLDN3”) was cloned as a receptor for CPE by Katahira et al. in 1997 (Non-patent Documents 2 and 3). At the same time, it has been reported that the gene of CLDN3 has a high homology with that of rat ventral prostate.1 protein (RVP.1) reported as a gene in which expression increases in rat prostate atrophy after castration (Non-patent Document 3). CLDN3 is a four-transmembrane protein containing total 220 amino acids in length and is one of the CLDN family constituting TJ.
Regarding expression of CLDN4 in cancers, its high level expression has been reported in various cancers such as ovarian cancer (Non-patent Documents 19, 20 and 21), breast cancer (N4 on-patent Document 22), uterine cancer (Non-patent Document 23), prostate cancer (Non-patent Documents 24 and 25), esophageal carcinoma (Non-patent Document 26), stomach cancer (on-patent Document 27) and colorectal cancer (Non-patent Document 28). Regarding the activity of CLDN3 in cancers, it has been reported that tumor forming ability and cell moving ability are accelerated in a CLDN3-expressed cancer cell and that the infiltrating ability of a cancer cell is decreased when expression of CLDN3 in the cell is suppressed using a small interfering RNA for CLDN3 (Non-patent Document 18). On the other hand, there are a report stating that expression of CLDN3 is decreased in a breast cancer cell line having high infiltrating ability (Non-patent Document 29) and a report stating that prognosis of CLDN3-positive patients of stomach cancer (Non-patent Document 30), so that there are many points which are still unclear regarding its function in cancers.
As a molecule which binds to CLDN3 and CLDN4, there is a Welch bacillus enterotoxin (CPE) which is a pathogenic bacterium of food poisoning. CPE is a membrane void-forming toxin which forms a complex by binding to CLDN3 or CLDN4 and destroys cells by cutting holes through the cell membrane. It has been reported that CPE has a cell-killing activity upon cancer cells which express CLDN3 or CLDN4, and shows the effect to suppress tumor growth or to prolong life (Non-patent Documents 9 and 10). Also, it has been reported that a C-terminal partial peptide of CPE (hereinafter referred to as “C-CPE”) from which the cell-killing activity of CPE was removed keeps its CLDN-binding activity, and inhibits the barrier activity of TJ by specifically suppressing expression of CLDN4 when it is added to a cell expressing CLDN4 (on-patent Document 4). Regarding C-CPE, activity of accelerating absorption via an intracellular space pathway (paracellular route) by inhibiting the barrier activity of TJ has also been reported (Non-patent Document 33).
As the antibody which binds to an extracellular region of CLDN4, a polyclonal antibody prepared by immunizing a domestic fowl with positions 141 to 158 in the amino acid sequence of CLDN4 represented by SEQ ID NO:2 and a monoclonal antibody prepared by immunizing a domestic fowl with positions 29 to 79 in the amino acid sequence of CLDN4 represented by SEQ ID NO:2 are known. It has been reported that the above-mentioned polyclonal antibody reacts with a cell expressing CLDN4 (Non-patent Document 34). In addition, as the above-mentioned monoclonal antibody, 4A4 (IgG3) is commercially available from Abnova. However, a monoclonal antibody which specifically recognizes three-dimensional structure of an extracellular region of CLDN4 and binds to the extracellar region and a monoclonal antibody which binds to an extraregion of CLDN4 and has neutralizing activity for CLDN4 have not so far been known.
As the antibody which binds to CLDN3, a rabbit polyclonal antibody which binds to mouse CLDN3 has been reported (Patent Document 1). In addition, regarding the antibody which recognizes an extracellular region of CLDN3 and binds thereto, it has been reported that a polyclonal antibody prepared by immunizing a domestic fowl with positions 56 to 69 and 139 to 156 in the amino acid sequence of CLDN3 represented by SEQ ID NO:26 react with a CLDN3-positive cancer cell (Non-patent Document 34). However, since the cancer cell to which the polyclonal antibodies has reactivity also expresses CLDN4, specificity of these antibodies has not been confirmed. Accordingly, a monoclonal antibody which specifically recognizes three-dimensional structure of an extracellular region of CLDN3 and binds thereto has not been known.
It is known that generally, when a non-human antibody such as a mouse antibody is administered to human, it is recognized as a foreign substance so that a human antibody for mouse antibody [human anti mouse antibody (HAMA)] is induced in the human body. It is known that HAMA reacts with the administered mouse antibody to thereby induce side effects (Non-patent Documents 35, 36, 37 and 38), quickens disappearance of the mouse antibody from the body (Non-patent Documents 36, 39 and 40) and decreases therapeutic effect of the mouse antibody (Non-patent Documents 41 and 42).
In order to solve these problems, attempts have been made to prepare a human chimeric antibody or a humanized antibody from a non-human antibody using gene recombination techniques.
A humanized antibody has various advantages in administration to human in comparison with a non-human antibody such as a mouse antibody. For example, it has been reported that its immunogenicity was decreased and its blood half-life was prolonged in a test using monkey, in comparison with a mouse antibody (Non-patent Documents 43 and 44). That is, since the humanized antibody causes fewer side effects in human than non-human antibodies, it is expected that its therapeutic effect is sustained for a prolonged time.
Also, since a humanized antibody is prepared using gene recombination techniques, it can be prepared as various forms of molecules. For example, when γ1 subclass is used as a heavy chain (hereinafter referred to as “H chain”) constant region (hereinafter referred to as “C region”) of a human antibody (H chain C region is referred to as “CH”), a humanized antibody having high effector functions such as antibody-dependent cellular cytotoxicity (hereinafter referred to as “ADCC activity”) can be prepared (Non-patent Document 43), and prolongation of its blood half life in comparison with mouse antibodies can be expected (on-patent Document 44), Particularly, in the case of treatment for suppressing proliferation of CLDN-positive cells, cytotoxic activities such as complement-dependent cytotoxicity (hereinafter referred to as “CDC activity”) via the Fc region (the region after the antibody heavy chain binge region) of an antibody and ADCC activity are important, in order to specifically damage the target cells by accumulating effector cells in a tumor tissue via the antibody. In the treatment of human, a human chimeric antibody, a humanized antibody or a human antibody is preferably used for exerting the cytotoxic activities (Non-patent Documents 45 and 46).
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